Immunofluorescence Studies
Six rabbits were used in this sub-study. The animals were anesthetized
with an overdose of pentobarbital sodium (30 mg/kg intravenously) and
anticoagulated with heparin (180 IU/kg). After median sternotomy, the
hearts were quickly excised. Then tissues were obtained from the SVC,
PVs, RA and LA, fixed in 4% paraformaldehyde until embedment in
paraffin. Four-micrometer sections were cut from paraffin blocks of
these tissues. The samples were incubated with primary antibodies
against c-TNT (1:100, Abcam, UK), KCa3.1 antibody
(Bioss, 1:200) overnight and then sections were subsequently with
secondary antibody, fluorescein isothiocyanate conjugated Affinipure
Goat Anti-rabbit IgG (Abcam, 1:50) and Fluorescein (CY3)-conjugated
Affinipure Goat Anti-mouse lgG (Aspen, 1:50). Cell nuclei was stained
with 4′,6-diamidino-2-phenylindole. Image Pro Plus 6.0 (Media
Cybernetics, Inc., Rockville, Maryland) was used to give quantitative
analysis. KCa3.1 expression in myocytes were calculated
by the sum of KCa3.1 density value in CTNT marked area.
Three visual fields at 400×magnification were then chosen randomly from
each sample to assessment.